LOW PASSAGE ASSOCIATED PROTEINS FORM B BURGDORFERI

低通道相关蛋白 B 型布格多菲

• 批准号: 2457759
• 负责人: FRANK C GHERARDINI
• 金额: $ 12.53万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-09-30 至 1999-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2457759
• 关键词: Borrelia; Lyme disease; bacterial proteins; bacterial vaccines; fusion gene; gene expression; genetic strain; hamsters; laboratory rabbit; molecular cloning; mutant; nucleic acid sequence; polymerase chain reaction; protein purification; recombinant proteins; transferrin; virulence

Outer surface proteins of Borrella burgdorferi that are essential for the bacterium to establish an infection would be excellent vaccine candidate for prevention of Lyme disease. Our laboratory has developed two methods for the identification and localization of these potential important proteins, We have developed: (1) an immune absorption technique; and (2) a membrane separation technique using equilibrium sucrose density centrifugation. With the first method, we identified ten proteins that are expressed only in low-passage, virulent B. burgdorferi strain B3I. With the second method, we have purified outer membranes of B. burgdorferL By combining these techniques, we have identified two (58- and 33-kDa) low- passage-associated outer membrane proteins. Both appear to be previously uncharacterized proteins and preliminary data suggests that one, the 58-kDa protein, may play a role in transferrin utilization by B. burgdorfen. In order to further characterize these proteins: (1) We will identify recombinant clones harboring genes encoding the 58-and 33-kDa low-passage- associated outer membrane proteins that we have previously identified. We will characterize positive clones using protein expression systems and sequence analysis. (2) We will begin the initial characterization of the 58 and 33-kDa proteins. We will construct gene fusions in order to purify recombinant 58- and 33-kDa proteins. Purified protein will be used for protection studies in mice. (3) We will isolate mutants of B. burgdorfeti that do not express low-passage-associated 58- and 33-kDa proteins and assess their role in virulence. Two recently described methods will be used: (a) the escape variant technique; and (b) gene disruption technique.

博氏疏螺旋体的外表面蛋白质，其对于 细菌建立感染将是很好的疫苗候选者 来预防莱姆病。我们的实验室开发了两种方法 为了识别和定位这些潜在的重要 我们已经开发了：（1）免疫吸收技术;和（2） 利用平衡蔗糖密度的膜分离技术 离心法用第一种方法，我们鉴定了10种蛋白质， 仅在低传代毒性B中表达。Burgdorferi菌株B3 I。与 第二种方法，我们纯化了B的外膜。BurgdorferL By 结合这些技术，我们已经确定了两个（58-和33-kDa）低- 与细胞膜相关的外膜蛋白。两人似乎都曾在 未表征的蛋白质和初步数据表明，一个，58-kDa的 转铁蛋白可能在B利用转铁蛋白中起作用。burgdorfen。在 为了进一步表征这些蛋白质：（1）我们将鉴定 携带编码58-和33-kDa低传代- 与我们之前鉴定的外膜蛋白相关。我们 将使用蛋白质表达系统表征阳性克隆， 序列分析(2)我们将开始对58 和33-kDa蛋白质。 我们将构建基因融合体， 重组58-和33-kDa蛋白。纯化蛋白将用于 在小鼠中的保护研究。(3)我们将分离出B的突变体。布格多尔费蒂 其不表达低传代相关的58-和33-kDa蛋白， 评估它们在毒性中的作用。最近描述的两种方法将是 使用：（a）逃逸变体技术;和（B）基因破坏技术。